1. Field of the Invention
The present invention relates to a process for the separation of a mixture of a protein and its reaction product with a polyalkylene glycol.
2. Description of the Related Art
When using therapeutic proteins in medicine, the proteins are frequently functionalized with a polyethylene glycol to prolong the biological half-life and thus the duration of action after the uptake into the patient's bloodstream. A frequently used synonym for a protein functionalized with a polyethylene glycol unit is the expression mono-, di-, or poly-“pegylated” protein where the protein molecule is optionally functionalized with one, two or more polyethylene glycol polymer chains. Owing to their greater hydrodynamic radius, pegylated proteins are eliminated more slowly via the kidneys than the corresponding nonpegylated proteins. Furthermore, the pegylation reduces a side-effect of the protein as antigen, which is undesired for the patient.
Efficient processes for the rapid determination of the amount of a mono-, di- and/or polypegylated protein in a substance mixture are becoming increasingly important in medical and biopharmaceutical rapid analysis because these processes provide within a short time reliable findings on the quantitative composition of such mixtures and on the degree of pegylation of the proteins present therein.
In recent years, a variety of processes have become established by means of which proteins, via their amino groups, can be reacted with chemically activated polyalkylene glycols, for example, polyalkylene glycols including aldehyde groups, to give Schiff's bases (imines), in which processes the imine groups of the protein are subsequently reduced to give pegylated amino groups. Depending on the number of amino groups of a protein which are involved in this reaction sequence with a polyethylene glycol, a mono-, di- or poly-“pegylated” protein results.
WO 2007/056191 A2 discloses a process for the purification of nucleotides which include a glycine unit whose amino group is functionalized with a polyethylene glycol unit. The monopegylated nucleotide can be separated from the unreacted pegylating reagent by a combination of membrane filtration (reverse osmosis or nanofiltration), size exclusion chromatography with polyacrylamide resins and ion-exchange chromatography with Q-Sepharose®. This document does not provide processes for the separation of mixtures which include mono-, di- and polypegylated nucleotides and/or proteins into the individual components.
WO 2008/154639 A2 discloses a process for the purification of monopegylated nucleotides in high yield and purity, where the monopegylated nucleotide is separated from the nonpegylated nucleotide and the unreacted pegylating reagent by anion-exchange chromatography using Q-Sepharose® gels, Mustang®-Q or Sartobind®-Q membrane adsorbers, followed by ultrafiltration and/or tangential flow filtration. Again, this document does not provide processes for the separation of mixtures which include mono-, di- and polypegylated nucleotides and/or proteins into the individual components.
WO 2006/011839 A1 discloses a process for the purification of a mono- or polypegylated 30 kDa protein which is not specified in more detail, using a chromatography gel based on a crosslinked copolymer of allyl dextran and N,N-methylenebisacrylamide, such as, for example, Sephacryl® S 500. Ion-exchanging or hydrophobic groups or affinity groups or metal-chelating groups are immobilized on the surface of the chromatography gel. The process allows the monopegylated protein to be separated from the polypegylated protein.
WO 2005/029065 A1 discloses chromatography matrices which are composed of crosslinked agarose on whose surface polyacrylic-acid-based polymer chains are fixed in place. Using these chromatography matrices, monopegylated proteins are separated from the starting materials of a pegylation reaction, i.e. from the unreacted pegylating reagent and from the nonpegylated proteins. It is the proton-donating carboxylic acid functions of the polyacrylic acid polymer chains which are essential to the success of the process. It has been found for the chromatography matrices disclosed in WO 2005/029065 A1 that, for a gradient elution, the binding interaction between the matrix and the components of the reaction mixture decreases in the order pegylating reagent>monopegylated component>nonpegylated component. As in the previously mentioned documents, this document, too, does not disclose any process for the separation of mono- and polypegylated-protein-containing mixtures into the individual components.
US 2005/0089952 A1 discloses the removal of a nonpegylated protein, for example lysozyme, from its mono- and polypegylated reaction products using membranes of polyether sulfone or regenerated cellulose in a tangential flow or diafiltration process. The decisive factor for the removal of the nonpegylated protein from the pegylated components is a molecular weight cut-off value (MWCO value) of the membranes of at least 30 kDa. Under this condition, the nonpegylated lysozyme passes across the membrane in the permeate stream while up to 97-99.2% of the pegylated lysozyme species are retained by the membrane. The aim of the separation process disclosed in US 2005/0089952 A1 is the efficient removal to the highest possible quantitative degree of the nonpegylated protein from the mono- or polypegylated protein species so that the nonpegylated protein may be recovered and recirculated into the reaction vessel for pegylation. Accordingly, this document, too, does not contain any disclosure as to how a mixture of mono- and polypegylated proteins can be separated into these species, using the abovementioned membranes.
In Biotechnol. Prog. 2007 (23), 1417-1424, J. R. Molek and L. Zydney disclose the removal of pegylated lactalbumin from nonpegylated lactalbumin and from further by-products by means of neutral or sulfonic-acid-group-functionalized ultrafiltration membranes based on regenerated cellulose and having an MWCO value of 30 or 100 kDa, respectively.
In their review paper from Chemical Engineering Science, 61, 2006, 924-939, C. J. Fee et al. disclose various separation methods, including size exclusion, affinity, hydrophobe interaction, cation exchange, metal chelate, reversed-phase and anion exchange chromatography methods for removing pegylated therapeutic proteins from their nonpegylated protein starting materials.